The long-term goal of this proposal is to understand, in detail, the mechanisms of mammalian mRNA 3' processing through comprehensive characterization of the 3' processing complex. mRNA 3'-end formation is an essential step of gene expression in eukaryotes, and has profound influences on many aspects of RNA metabolism, including mRNA stability, export, and translation. Additionally, regulated mRNA 3' processing has emerged as a critical mechanism for gene control. Aberrant 3' processing signals and mutant 3' processing factors cause a number of human diseases, including cancer. For almost all eukaryotic mRNAs, 3' processing involves two catalytic steps, an endonucleolytic cleavage followed by the addition of a poly(A) tail. Both of these steps take place in a macromolecular machinery, the 3' processing complex. The structure-function relationship of this complex is a central question in understanding the mechanism of mRNA 3' processing. We have recently purified the functional 3' processing complex assembled on its mRNA target and initiated proteomic, functional, and structural analyses. Building on our purification system, we will address several fundamental and long-standing questions in the field: How are the 3' processing signals in mRNAs specifically recognized? What is the protein composition of the 3' processing complex and (how) does it change during 3' processing reaction? What is the structure of the 3' processing complex? To achieve our objectives, we have designed the following specific aims: 1. Comprehensively map the protein-RNA interactions within the purified 3' processing complex. 2. Characterize the compositional changes of the 3' processing complex during 3' processing reaction. 3. Characterize the structural changes of the 3' processing complex during 3' processing reaction using single particle cryo-electron microscopy (cryo-EM). Accomplishment of the proposed studies will provide new and significant mechanistic insights into the structure-function relationship and the dynamics of the mRNA 3' processing complex, and may also provide the foundation for development of new therapeutic approaches against diseases caused by aberrant 3' processing.